Metabolic engineering has allowed production of chemicals of commercial interest through manipulation of biochemical reactions in the cell. However, all organisms have evolved with the objective of replicating their genetic material and, therefore, production of chemicals that may be of commercial interest may conflict with essential cellular goals. For example, diversion of nutrients and energy for the production of a compound may result in a shortage of those substrates for production of biomass. The organism that is engineered may either evolve away from producing the compound of interest or grow sub-optimally.
To address this issue, systems have been engineered for stationary phase-associated production of a compound of interest. However, a limitation is that stationary phase itself elicits a series of responses that are aimed at protecting the cell during non-growing conditions, so cellular resources are still needed for combating stress and preserving the stationary-phase phenotype. This effect may be pronounced when the product of interest is itself toxic or induces a stress response.
Recently, the development of cell-free systems has allowed for the in vitro production of proteins (for a review see Swartz (2006) J Ind Microbiol Biotechnol 33:476-85). A cell-free platform may also be used to produce metabolites of interest through coordinated expression of proteins in a pathway. The procedure entails growing a biomass of cells, opening the cells to liberate the cytoplasmic components, removing the genomic DNA, and using the genome-free machinery to produce a user-specified set of enzymes to serve as biocatalysts. This presents a unique opportunity for producing an environment that resembles stationary phase production systems but free of stress or other responses, although a limitation may be relatively slow kinetics of the set of reactions, given that diffusion of intermediates from one enzyme into the next is needed for a pathway to work.